[NaCI i

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Nanodiamond Doxorubicin

Figure 8.17. Schematic diagram of NaCl-mediated loading and releasing of DOX with NDs. Source: From Ref. 18.

resulted in maintaining the protein conformations allowing better antibody binding and hence a strong immune response.

For the first time, Huang and coworkers18 demonstrated that NDs serve as efficient chemotherapeutic drug carriers. Doxorubicin hydrochloride (DOX), an apoptosis-inducing drug used in chemotherapy, was coated on the NDs or embedded into the intervals of ND aggregates and introduced into living cell. NDs moved quickly inside cell and served as a stabilization matrix for DOX to preserve drug functionality. First, the concentration of NaCl was found to be able to control the absorption/desorption of DOX onto ND as shown in Fig. 8.17. Then, the interaction between DOX/ND and cell was studied. NDs were also coated with fluorescent poly-L-lysine. Figure 8.18 shows that the NDs are attached to cell instantly and interact with the living cells dynamically. Within 10 h, NDs are internalized in the cell.

8.5 BIOMARKERS AND BIOLABELING

As in Fig. 8.3, very strong fluorescence at 700 nm is emitted after excited at 560 nm20 due to the nitrogen-vacancy (N-V)- center within diamonds. This is advantageous for imaging in biological cells, as the background fluorescence in cell is 300-400 nm.5 Fu and coworkers found that under the same excitation conditions,21 the

Figure 8.18. (a-e) Confocal images of macrophage RAW cells with addition of NDs (20 ^g/ml) coated with fluorescent poly-L-lysine (FTTC-PL/ND ~10 wt%). (a,b) Taken from the cells after the addition of NDs without and with incubation (37°C for 10 h), respectively. The excitation wavelength is 488 nm. (d,e) are bright-field images corresponding to (a), (b), respectively. (c) From the same cells as in (b) but was stained with DNA-binding dye TOTO-1 and excited with 514 nm. The nucleus of the cell can be clearly identified. (f) TEM image showing NDs in macrophage cytoplasm. The image was taken after 3 h incubation of the cells with addition of DOX coated NDs (10wt%). The cells are dehydrated, fixed, and sliced for TEM observation. The scale is 20 nm. Source: From Ref. 18.

Figure 8.18. (a-e) Confocal images of macrophage RAW cells with addition of NDs (20 ^g/ml) coated with fluorescent poly-L-lysine (FTTC-PL/ND ~10 wt%). (a,b) Taken from the cells after the addition of NDs without and with incubation (37°C for 10 h), respectively. The excitation wavelength is 488 nm. (d,e) are bright-field images corresponding to (a), (b), respectively. (c) From the same cells as in (b) but was stained with DNA-binding dye TOTO-1 and excited with 514 nm. The nucleus of the cell can be clearly identified. (f) TEM image showing NDs in macrophage cytoplasm. The image was taken after 3 h incubation of the cells with addition of DOX coated NDs (10wt%). The cells are dehydrated, fixed, and sliced for TEM observation. The scale is 20 nm. Source: From Ref. 18.

fluorescence of a single 35 nm diamond is significantly brighter than that of a single dye molecule such as Alexa Fluor 546.

Fu and coworkers21 used fluorescent nanodiamond (FND) coated with ploy-L-lysine (PL) to study the interaction between DNA and FND on an amine-terminated glass substrate. The PL was used to facilitate the binding of DNA (fluorescently labeled with TOTO-1 dye molecule) to FND. Due to the specific strong fluorescence at 700 nm of FND, Fig. 8.19 clearly shows that the DNA molecule is wrapped around the PL-coated FND particle. Fu and coworkers21 also demonstrated that it is possible to conduct a single particle tracking for a 35-nm FND in the cytoplasm of a live Hela cell. Figure 8.20 shows the diffusion of FND in the Hela cells. The bright red dot in (A) is the FND, whereas (B) shows the trajectory of FND starting from the origin (0 0). This tracking method

545 -6D5 nm

Figure 8.19. Observation of a single FND particle bound with a single T4 DNA molecule on an amine-terminated glass substrate. Dual-view fluorescence images of a single DNA/FND complex. An overlay (Right) of the images (545-605 nm) and longer (675-685 nm) wavelength channel reveals that Ta DNA was wrapped around the 100 nm FND particle and stretched to a V-shape configuration. Source: From Ref. 21.

Figure 8.19. Observation of a single FND particle bound with a single T4 DNA molecule on an amine-terminated glass substrate. Dual-view fluorescence images of a single DNA/FND complex. An overlay (Right) of the images (545-605 nm) and longer (675-685 nm) wavelength channel reveals that Ta DNA was wrapped around the 100 nm FND particle and stretched to a V-shape configuration. Source: From Ref. 21.

B 300

Figure 8.20. Tracking of a single FND in a live Hela cell. (A) Overlay of bright-field and epifluorescence images of a live Hela cell after an uptake of 35-nm FNDs. (B) A 100-step (139 ms per step) trajectory of a single FND, indicated by the yellow in A, moving in the cytoplasm of the Hela cell. Source: From Ref. 21.

B 300

-400

-400

-400 0 400 X (nm)

Figure 8.20. Tracking of a single FND in a live Hela cell. (A) Overlay of bright-field and epifluorescence images of a live Hela cell after an uptake of 35-nm FNDs. (B) A 100-step (139 ms per step) trajectory of a single FND, indicated by the yellow in A, moving in the cytoplasm of the Hela cell. Source: From Ref. 21.

could be applied to drug delivery system of ND, where the interaction between ND and cell could be monitored using fluorescence microscopy.

Raman spectrum of diamond exhibits a sharp peak at 1332 cm-1. And the peak is isolated and the Raman absorption cross section is large.19 This peak can be used as an indicator of the location of nanodiamond. Cheng and co-workers22 tracked growth hormone factor in one single cancer cell using nanodiamond-growth hormone complex (cND-rEaGH) as a specific probe. The

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Figure 8.21. (A) Confocal Raman mapping image of A549 cell and 100 cND. (B) Color Raman mapping of A549 cell and 100 cND-rEaGH carboxylated nanodiamond complexes. The images shown are at different z position scans: (a) at z = 10 ^m with diamond collected in 13201340 cm-1 and cell collected in 1432-1472 cm-1; (b) at z = 0 ^m position; (c) at z = —10 ^m. Source: From Ref. 25.

Figure 8.21. (A) Confocal Raman mapping image of A549 cell and 100 cND. (B) Color Raman mapping of A549 cell and 100 cND-rEaGH carboxylated nanodiamond complexes. The images shown are at different z position scans: (a) at z = 10 ^m with diamond collected in 13201340 cm-1 and cell collected in 1432-1472 cm-1; (b) at z = 0 ^m position; (c) at z = —10 ^m. Source: From Ref. 25.

growth hormone factor of A549 human lung epithelial cell can recognize the growth hormone. Using Raman mapping, the endocy-tosis of cND and cND-rEaGH were observed (shown in Fig. 8.21). From (A), cND can penetrate inside cells, whereas from (B), cND-rEaGH resides only on the surface of the cell. This observation is consistent with the fact that hormone-binding domain of growth hormone receptor is extracellular domain23 and the GH/GHR complex forming is on the extracellular part of membrane. Using the same method, Chao studied the interaction of E. Coli with cND-lysozyme complex.24 The lysozyme can be attached to a carboxy-lated ND through physical absorption.25 Figure 8.22 shows that the cND can be used as a marker to identify the location of the interacting protein lysozyme with E. Coli using Raman mapping.24

8.6 ELECTROCHEMICAL APPLICATIONS

Due to its physicochemical stability, large electrochemical potential winow, and chemical sensitivity,27 diamond is an excellent candidate for electrochemical applications. Diamond electrodes show the most stable response among electrodes by far, and do

E. coli alone

E. coli alone

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Raman signal (100 cND)

optical ¡image

Raman signal (100 cND)

Figure 8.22. The interaction of E. Coli with cND-lysozyme complex as viewed with a conventional optical microscope (objective 100x) and con-focal Raman spectrometer. The location of the cND is indicated in yellow. Arrow points to the location of cND. Source: From Ref. 24.

not require extensive pretreatment to regenerate the electroactive surface.28 Diamond electrodes/microelectrodes have been applied to biological system as biosensors.29

Rubio-Retama studied the nanocrystalline diamond (NCD) electrodes immobilized with horseradish peroxidase (HRP),30 which reacts with H2O2 through reduction/oxidation (redox) reaction, and found that using the modified electrode for hydrogen peroxide determination shows a linear response in the 0.1-45 mM H2O2 range. Using the same immobilization method as Yang,8 HRP was immobilized to the NCD electrode surface. Figure 8.23 shows the model of modified NCD structure.30 The modified NCD electrode as biosensor showed a linear response in the 0.1-45 mM H2O2 range, at +0.05 V vs Ag/AgCl, for hydrogen peroxide determination. The enzymatic activity was constant during the stability study

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